Schelhas, C. | bioRxiv Science

bioRxiv2026

Split-Indigoidine synthetase as optical reporter for benchmarking protein-protein interactions

Indigoidine is a blue pigment biosynthesized by a single-module Non-Ribosomal Peptide Synthetase (NRPS) using L-glutamine as substrate. Despite its potential as a colorimetric reporter, no such system has been established from it to date. We used a recently characterized interdomain fusion site located between its adenylation (A) and thiolation (T) domains to develop the Indi2GO system, which provides a naked-eye detectable and quantitative optical readout of transient and covalent protein-protein-interaction (PPI) in living cells. Indi2GO enables high-throughput benchmarking and optimization of PPI tools in a standard 96-well plate reader format, without requiring exogenous substrates, specialized equipment or complex analytical workflows. We demonstrate its broad applicability with three widely used protein-protein interaction tools: SYNZIPS, inteins, and the SpyTag:SpyCatcher system. We used Indi2GO to validate novel SYNZIP pairs, which we used in NRPS engineering, highlighting its applicability for the development of novel PPI-mediating tools in the context of NRPS engineering and synthetic biology.

synthetic biology↗

bioRxiv2025

Split inteins for generating combinatorial non-ribosomal peptide libraries

Engineering Non-Ribosomal Peptide Synthetases (NRPS) is a promising strategy for discovering new bioactive compounds, which can serve as valuable leads for drug development, such as new antibiotics. However, their engineering is hampered by the limited availability of molecular tools for the efficient heterologous expression of their large biosynthetic gene clusters. In fact, a single NRPS gene can already exceed the size limits of standard cloning vectors. In this study, we establish split inteins as a novel tool for NRPS engineering to enable the expression of single, covalently linked NRPS proteins from multiple plasmids and to perform cloning-free module swapping. Using the xenotetrapeptide synthetase as model system, we show that an NRPS can be split into three parts and reconstituted via trans-splicing using two orthogonal inteins at four different engineering sites. Based on this tripartite platform we build a library comprising 21 plasmids and generated 324 hybrid NRPS by combinatorial transformation. More than half were catalytically active, producing over 200 novel peptides. This intein-based technology provides a modular platform for generating natural product-like peptide libraries, expanding biocatalytically accessible chemical space.

synthetic biology↗